Abstract
The aryl hydrocarbon receptor (AhR) is a transcription factor that induces the adaptive responses upon binding to a wide range of exogenous chemicals in cells. The traditional method for studying AhR ligands is to determine their effect on transcriptional activities (e.g., luciferase expression) in cultured cells. In this paper, we sought to investigate the feasibility of studying the ligand binding with the AhR using a Förster resonance energy transfer (FRET) assay-a novel approach. Two conjugates (βNFQ and 8) containing β-naphthoflavone and DABCYL (a quencher) with different linkers were therefore synthesized for evaluation. The luciferase expression and green fluorescent protein (GFP) expression assays showed that βNFQ is a partial AhR agonist. Simultaneous incubation of cultured cells containing expressed GFP-AhR fusion protein with βNFQ and a known AhR ligand 3-methylchloranthrene reduced the fluorescence of GFP-AhR to a lesser extent, suggesting that the reduction in fluorescence resulted from the competitive binding of ligands to GFP-AhR. Although the fluorescent quenching by βNFQ is modest, our results suggest that FRET could be a valuable tool for identifying AhR ligands with the use of a more efficient ligand-quencher.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 711-721 |
| Number of pages | 11 |
| Journal | Medicinal Chemistry Research |
| Volume | 21 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2012 |
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Keywords
- β-Naphthoflavone
- Aryl hydrocarbon receptor ligands
- Förster resonance energy transfer
- Green fluorescence protein
ASJC Scopus subject areas
- Pharmacology, Toxicology and Pharmaceutics(all)
- Organic Chemistry
Cite this
Synthesis of a ligand-quencher conjugate for the ligand binding study of the aryl hydrocarbon receptor using a FRET assay. / Wang, Yu; Yang, Dazhou; Chang, Abraham; Chan, William K.; Zhao, Bin; Denison, Michael S.; Xue, Liang.
In: Medicinal Chemistry Research, Vol. 21, No. 6, 06.2012, p. 711-721.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Synthesis of a ligand-quencher conjugate for the ligand binding study of the aryl hydrocarbon receptor using a FRET assay
AU - Wang, Yu
AU - Yang, Dazhou
AU - Chang, Abraham
AU - Chan, William K.
AU - Zhao, Bin
AU - Denison, Michael S.
AU - Xue, Liang
PY - 2012/6
Y1 - 2012/6
N2 - The aryl hydrocarbon receptor (AhR) is a transcription factor that induces the adaptive responses upon binding to a wide range of exogenous chemicals in cells. The traditional method for studying AhR ligands is to determine their effect on transcriptional activities (e.g., luciferase expression) in cultured cells. In this paper, we sought to investigate the feasibility of studying the ligand binding with the AhR using a Förster resonance energy transfer (FRET) assay-a novel approach. Two conjugates (βNFQ and 8) containing β-naphthoflavone and DABCYL (a quencher) with different linkers were therefore synthesized for evaluation. The luciferase expression and green fluorescent protein (GFP) expression assays showed that βNFQ is a partial AhR agonist. Simultaneous incubation of cultured cells containing expressed GFP-AhR fusion protein with βNFQ and a known AhR ligand 3-methylchloranthrene reduced the fluorescence of GFP-AhR to a lesser extent, suggesting that the reduction in fluorescence resulted from the competitive binding of ligands to GFP-AhR. Although the fluorescent quenching by βNFQ is modest, our results suggest that FRET could be a valuable tool for identifying AhR ligands with the use of a more efficient ligand-quencher.
AB - The aryl hydrocarbon receptor (AhR) is a transcription factor that induces the adaptive responses upon binding to a wide range of exogenous chemicals in cells. The traditional method for studying AhR ligands is to determine their effect on transcriptional activities (e.g., luciferase expression) in cultured cells. In this paper, we sought to investigate the feasibility of studying the ligand binding with the AhR using a Förster resonance energy transfer (FRET) assay-a novel approach. Two conjugates (βNFQ and 8) containing β-naphthoflavone and DABCYL (a quencher) with different linkers were therefore synthesized for evaluation. The luciferase expression and green fluorescent protein (GFP) expression assays showed that βNFQ is a partial AhR agonist. Simultaneous incubation of cultured cells containing expressed GFP-AhR fusion protein with βNFQ and a known AhR ligand 3-methylchloranthrene reduced the fluorescence of GFP-AhR to a lesser extent, suggesting that the reduction in fluorescence resulted from the competitive binding of ligands to GFP-AhR. Although the fluorescent quenching by βNFQ is modest, our results suggest that FRET could be a valuable tool for identifying AhR ligands with the use of a more efficient ligand-quencher.
KW - β-Naphthoflavone
KW - Aryl hydrocarbon receptor ligands
KW - Förster resonance energy transfer
KW - Green fluorescence protein
UR - http://www.scopus.com/inward/record.url?scp=84861901924&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84861901924&partnerID=8YFLogxK
U2 - 10.1007/s00044-011-9575-7
DO - 10.1007/s00044-011-9575-7
M3 - Article
AN - SCOPUS:84861901924
VL - 21
SP - 711
EP - 721
JO - Medicinal Chemistry Research
JF - Medicinal Chemistry Research
SN - 1054-2523
IS - 6
ER -